Structural basis of neuropeptide Y signaling through Y1 and Y2 receptors

Abstract Neuropeptide Y (NPY), a 36‐amino‐acid peptide, functions as a neurotransmitter in both the central and peripheral nervous systems by activating the NPY receptor subfamily. Notably, NPY analogs display varying selectivity and exert diverse physiological effects through their interactions with this receptor family. [Pro34]–NPY and [Leu31, Pro34]–NPY, mainly acting on Y1R, reportedly increases blood pressure and postsynaptically potentiates the effect of other vasoactive substances above all, while N‐terminal cleaved NPY variants in human body primary mediates angiogenesis and neurotransmitter release inhibition through Y2R. However, the recognition mechanisms of Y1R and Y2R with specific agonists remain elusive, thereby hindering subtype receptor‐selective drug development. In this study, we report three cryo‐electron microscopy (cryo‐EM) structures of Gi2‐coupled Y1R and Y2R in complexes with NPY, as well as Y1R bound to a selective agonist [Leu31, Pro34]–NPY. Combined with cell‐based assays, our study not only reveals the conserved peptide‐binding mode of NPY receptors but also identifies an additional sub‐pocket that confers ligand selectivity. Moreover, our analysis of Y1R evolutionary dynamics suggests that this sub‐pocket has undergone functional adaptive evolution across different species. Collectively, our findings shed light on the molecular underpinnings of neuropeptide recognition and receptor activation, and they present a promising avenue for the design of selective drugs targeting the NPY receptor family.


INTRODUCTION
2][3] They can be activated by three structurally related, but functionally diverse endogenous peptides: NPY, peptide YY, and pancreatic polypeptide (PP). 4,5All three peptides comprise 36 amino acids each with amidated C-terminal ends and share highly conserved amino acid sequences. 6,7Among them, NPY could specifically activate Y 1 R, Y 2 R, and Y 5 R, and it is broadly distributed in the central and peripheral nervous system, regulating various physiological processes such as food intake, stress response, anxiety, and memory retention. 8,9PY is cleaved into two N-terminal truncated NPY variants during cellular metabolism: NPY 3−36 and NPY 2−36 , processed by dipeptidyl peptidase IV and aminopeptidase P (AmP), respectively.[10][11][12][13] The truncated NPY peptides lose their binding affinity for Y 1 R, but they retain a similar binding affinity with Y 2 R. 14,15 In addition, the synthesized NPY analog [Leu 31 , Pro 34 ]-NPY is reported to be specific agonist at Y 1 R, sharing similar activation potency with NPY for Y 1 R. [16][17][18][19] Y 1 R and Y 2 R are known to be primarily expressed at the post-and presynaptic membranes, respectively.[20][21][22] [Pro 34 ]-NPY and [Leu 31 , Pro 34 ]-NPY, when acting on Y 1 R, primarily raise blood pressure and postsynaptically potentiate the effects of other vasoactive substances.23][24][25][26][27] These results suggest that Y 1 R and Y 2 R exhibit different ligand recognition mechanisms and could potentially allow for selective drug development for corresponding condition. Hoever, selective agonist development would require the identification of the underlying mechanisms of receptor-peptide ligand interactions. Although structural information of Y 1 R-and Y 2 R has been reported, [28][29][30] the underlying ligand selectivity and receptor activation mechanisms remain unclear.Recently, structural studies on the NPY 1 R have elucidated the molecular mechanisms underlying Y 1 R recognition of NPY.However, the molecular basis for why Y 1 R requires the complete N-terminus of NPY for optimal activation and why [Leu 31 , Pro 34 ]-NPY selectively activate Y 1 R rather than Y 2 R remains unclear.Our study has provided new insights to address these mentioned issues.
In this study, we present three single-particle cryoelectron microscopy (cryo-EM) structures of the Y 1 R/Y 2 R-Gi2 signaling complexes bound to endogenous peptide NPY, as well as Y 1 R-Gi2 in complex with a selective ligand [Leu 31 , Pro 34 ]-NPY.Moreover, we performed structural comparison and identified a unique sub-pocket in Y 1 R that accommodates the N-terminal NPY residues.Further, the results of our adaptive evolution analysis demonstrated that positive Darwinian selection occurred specifically in Y 1 R from Osteichthyes.In addition, our structural analysis reveals that the residue Pro 34 substitution in NPY specifically interrupted the extensive NPY-Y 2 R interaction but is more compatible with Y 1 R. Our study elucidates key residues required for NPY peptide recognition and deciphers the plasticity of orthosteric site of NPY receptor subtypes in response to the same ligand stimuli.

Cryo-EM structures of Y 1 R/Y 2 R-Gi2 signaling complexes
In order to investigate Y 1 R and Y 2 R activation potency in response to NPY variants, including N-terminally truncated NPY and NPY mutants (Figure 1A), we measured the cAMP inhibition ability of wildtype Y 1 R and Y 2 R elicited by different agonists in HEK293 cells (Figures 1A-C).Our results indicated that deleting the N-terminal residues     -NPY, NPY  , and NPY  , respectively. Valus are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate. (C) Cncentration-response curves of Y 2 R in response to stimulation with NPY, [Leu 31 , Pro 34 ]-NPY, NPY  , and NPY  , respectively.Values are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate.(D) Orthogonal views of the electronic density map of the Y 1 R-NPY-Gi2-complex.The Y 1 R and NPY are colored cyan and orange, respectively; and Gαi 2 , Gβ, Gγ, and scFv16 are colored tan, dark cyan, (Y 1 and P 2 ) of NPY (NPY 3−36 ) and NPY 13−36 remarkably reduced Y 1 R activation compared to NPY (>50 folds), whereas [Leu 31 , Pro 34 ]-NPY analog retained a potency nearly identical to NPY (Figure 1B).In contrast, the NPY 3−36 variant exhibited slightly reduced Y 2 R activation and [Leu 31 , Pro 34 ]-NPY was ∼100-fold less potent than NPY on Y 2 R activation (Figure 1C).These observations suggest that Y 1 R and Y 2 R display different ligand recognition mechanisms.
To gain a more thorough understanding of how Y 1 R and Y 2 R recognize NPY or NPY mutants, we determined the structures of NPY-bound Y 1 R, NPY-bound Y 2 R, and [Leu 31 , Pro 34 ]-NPY-bound Y 1 R in complex with the Gi2 protein using cryo-EM single-particle technique (Figures 1D-I and S1 and 2, and Table S1).The final cryo-EM maps of NPY-Y 1 R-Gi2 (PDB ID:8K6M), NPY-Y 2 R-Gi2 (PDB ID:8K6N), and [Leu 31 , Pro 34 ]-NPY-Y 1 R-Gi2 (PDB ID: 8K6O) displayed global nominal resolutions of 3.5, 3.2, and 3.3 Å after refinement, respectively (Figures S1 and 2 and Table S1).The Y 1 R and Y 2 R structure shared a canonical seven-transmembrane (TM) helical architecture and an intracellular amphipathic helix 8.The seven TM helix domains of both receptors could be visibly distinguished in the cryo-EM maps (Figure S3), and the high-resolution density maps allow us to unambiguously build the NPY, [Leu 31 , Pro 34 ]-NPY, NPYRs, Gαi 2 , Gβ, Gγ, and scFv16 (Figures 1D-I and S3).However, probably due to the NPY flexibility in the extracellular region, we failed to model the residues 10−15 from NPY and [Leu 31 , Pro 34 ]-NPY and the residues 10−14 from NPY in Y 1 R and Y 2 R complex structures, respectively (Figures 1G-I).
By comparing NPY-Y 1 R or NPY-Y 2 R with their inactive states (PDB ID: 5ZBQ and 7VGX, respectively), 31,32 we noticed that the intracellular end of TM6 in two receptors exhibited an obvious outward shift to accommodate the Gαi 2 α5 helix and TM7 also adopted an inward displacement similar to other active class A GPCR structures [33][34][35] (Figures S4A and B).As the NPY-Y 1 R and [Leu 31 , Pro 34 ]-NPY-Y 1 R structures share highly similar conformation with a Cα root mean square deviation (RMSD) of 0.569 Å (279-279 atoms), we use the NPY-Y 1 R-Gi2 and NPY-Y 2 R-Gi2 structures to analyze the overall Y 1 R and Y 2 R structures in this section (Figure S4C).
Although Y 1 R and Y 2 R shared only 30% sequence identity, these two receptors activation complex structures resembled a similar conformation with a Cα RMSD of 0.905 Å (213-213 atoms) (Figure S4D).It is noteworthy that the extracellular end of TM1, TM4, and TM5 in Y 1 R exhibited a slight outward movement relative to that in Y 2 R (Figure S4D).The ligand NPY displayed similar binding pattern both in the Y 1 R and Y 2 R structures and folded into a canonical PP-fold, 36 the C-terminal regions (residues 32−36) were inserted into an orthosteric pocket of Y 1 R and Y 2 R, while the N-terminals (residues 1−10) folded back to form extensive interactions with the NPY α-helix (residues 15−31) (Figures 1G-H and S5A-C).

A sub-pocket in Y 1 R determines NPY selectivity
The NPY-Y 1 R with NPY-Y 2 R structural comparison revealed remarkable displacements (∼9 Å) in the NPY N-terminal part (Figure S5D).In Y 1 R, the NPY N-terminal end could be found in a cavity composed of the extracellular ends of TM5, TM6, and ECL2.However, the NPY N-terminal part in Y 2 R extended toward the ECL2 region, forming relatively few contacts with the receptor.In accordance with the NPY pharmacological assay, the structure revealed different NPY recognition mechanisms in Y 1 R and Y 2 R.
In detail, the NPY N-terminal Y 1 and P 2 residues fitted into the amphiphilic cavity formed by hydrophobic (P183 ECL2 , F199 ECL2 , and F286 6.58 ) and polar (E182 ECL2 and D200 ECL2 ) residues, and the corresponding [Leu 31 , Pro 34 ]-NPY residue adopted a conformation similar to that of NPY (Figure S6A).Therefore, we defined the cavity in Y 1 R as a unique sub-pocket (Figures 2A and B), where the clusters of hydrophobic residues (P183 ECL2 , F199 ECL2 , and F286 6.58 ) formed a hydrophobic contact network with Y 1 and P 2 residues and D200 ECL2 and E182 ECL2 established a polar network with the NPY peptide Y 1 and K 4 side chains (Figure 2B).In addition, two critical residues R208 6.35 and F286 6.58 bifurcated the NPY binding pocket in Y 1 R into two cavities, a sub-pocket, and a major orthosteric pocket (Figure 2B).In contrast to the Y 1 R NPY recognition mode, Y 2 R did not contain the equivalent sub-pocket for NPY binding (Figures 2C and D).
To further identify the key residues of the Y 1 R subpocket for the NPY N-terminus recognition, we conducted a series of mutagenesis analyses.The Y 1 R and Y 2 R sequence alignment results confirmed that the Y 1 R pink and silver.(E) Orthogonal views of the electronic density map of the Y 2 R-NPY-Gi2-complex.The Y 2 R and NPY are colored medium slate blue and magenta, respectively; and Gαi, Gβ, Gγ, and scFv16 are colored tan, dark cyan, pink, and silver.(F) Orthogonal views of the electronic density map of the Y 1 R-[Leu 31 , Pro 34 ]-NPY-Gi2-complex.The Y 1 R and [Leu 31 , Pro 34 ]-NPY are colored medium blue and light salmon, respectively; and Gαi, Gβ, Gγ, and scFv16 are colored tan, dark cyan, pink, and silver.(G -I ) Ribbon representation of the 31 , Pro 34 ]-NPY-Gi2 complexes, colored according to Cryo-EM maps.sub-pocket residues (E182 ECL2 , D200 ECL2 , P183 ECL2 , F199 ECL2 , and F286 6.58 ) were not conserved (Figure 2E).We replaced these key residues with the corresponding residues in Y 2 R and our mutagenesis test demonstrated that mutations E182I, D205S, and H290S in Y 1 R did not affect the receptor activation potency (Figure 2F and Table S2).However, the hydrophobic residues of subpocket mutations (P183 ECL2 E, F199 ECL2 T, F286 6.58 V, and P183 ECL2 E/F199 ECL2 T/F286 6.58 V) all severely reduced Y 1 R potency to NPY (Figure 2G and Table S2).Our results indicate that the sub-pocket-forming hydrophobic residues contribute to the Y 1 R-mediated NPY recognition.
Compared with other β-branch structures of class A GPCRs, for instance, the endogenous endothelin ET-1 peptide-bound ET B receptor structure revealed a similar peptide-receptor binding mechanism as that in Y 1 R, in which the extracellular part of the receptor (ECL2, TM6, and ECL3) is widely involved in the N-terminal ET-1 binding 33 (Figure S6B).However, the N-terminal ET-1 region mainly forms hydrogen bonds and electrostatic interactions with ET-1 and no such Y 1 R sub-pocket has been observed in the ET B -ET1 structure (Figures S6B-C).
Our structures revealed a sub-pocket, which plays important roles in the N-terminal NPY recognition and receptor activation of Y 1 R. Our observation was consistent with previous Y 1 R-related NPY NMR modeling and docking studies. 37Taken together, our study provides insights into the detailed NPY N-terminus-Y 1 R interactions and helps in fully understanding the different selectivity mechanisms of various N-terminally truncated NPY peptides upon Y 1 R and Y 2 R activation.

2.3
Adaptive evolution of the Y 1 R sub-pocket NPY and Y 1 R evolutionary dynamics in early vertebrates have already been thoroughly studied. 2,38However, the evolutionary fate of Y 1 R sub-pocket is elusive.We collected 22 genomes of vertebrates and invertebrates to reveal the related evolutionary processes.Our results revealed that NPY and Y 1 R were present in vertebrates such as Cyclostomata, Chondrichthyes, Osteichthyes, and Tetrapoda, but not in invertebrates (Figure 3A).We selected six classical vertebrate Y1Rs for signal pathway detection and the results demonstrated that Cyclostomata (pmY 1 R/Petromyzon marinus), Osteichthyes (drY 1 R/Danio rerio), and Tetrapoda (hY 1 R/Homo Sapiens, and cpbY 1 R/Chrysemys picta bellii) Y1Rs could be activated by NPY, while such activation is significantly weakened (scY 1 R EC 50 = 175.1 nM and ccY 1 R could not be activated by NPY) in Chondrichthyes (scY 1 R/Scyliorhinus canicula and ccY 1 R/Carcharodon carcharias) (Figures 3A and B).The branch-site model has proved to be a useful tool for detecting biological hypotheses of positive selection and generative mutation research and functional analysis. 39o, we used a branch-site model to assess whether the Y 1 Rs underwent positive selection. 40,41As shown in Figure 3C, the Y 1 R sequences exhibited a relevant nonsynonymous (dN)/synonymous (dS) substitution rate ratio (branch-site dN/dS of ω > 1; Figures 3A and C) that was highly significant (likelihood ratio tests [LRTs], p < 0.05; Figure 3C) only in the Osteichthyes lineage.In contrast, Chondrichthyes did not exhibit this ratio, suggesting that positive Darwinian selection occurred specifically in Osteichthyes, but not in Chondrichthyes Y 1 Rs (Figure 3C).And we found that the sub-pocket residues Q182 and R203 in Osteichthyes Y 1 R were under positive selection (Figure 3C).Furthermore, according to sequence logos between Osteichthyes and Chondrichthyes Y 1 Rs, the six sub-pocket residues were non-conserved (Figure 3D).
To verify the Y 1 R-related functional differences between Chondrichthyes and other vertebrates, three vertebrate species (human/Homo Sapiens, zebrafish/Danio rerio, and Carcharodon/Carcharodon carcharias representing Tetrapoda, Osteichthyes, and Chondrichthyes, respectively) were selected to test the biological experimental research (Figure 3E).We used the NPY peptide to activate the different Y 1 Rs.NPY activated humans and zebrafish, but not Carcharodon, Y 1 Rs.To further investigate the function of the six residues (E182 ECL2 , Q186 ECL2 , F199 ECL2 , D200 ECL2 , D205 5.32 , F286 6.58 , and H290 ECL3 ) in the subpocket, the human Y 1 R residues (hY 1 R) were mutated to the corresponding residues in the Chondrichthyes orthologs.Similar to the effect of mutating the corresponding key residues in Y 2 R, residue F199 ECL2 and F286 6.58 substitutions markedly affected the hY 1 R activation efficacy in response to the NPY peptide (Figure 3E).In addition, residues N186 and D205 in human Y 1 R sub-pocket under positive selection were also engaged in NPY recognition (Figure S7A).Next, Carcharodon Y 1 R (ccY 1 R) (Q181 ECL2 , Y184 ECL2 , I199 ECL2 , E205 5.32 , and Y290 ECL3 ) and zebrafish Y 1 R (drY 1 R) (A188 ECL2 , Q192 ECL2 , V208 ECL2 , R214 5.32 , and I295 6.58 ) residues were reversely mutated to the corresponding hY 1 R.We observed that mutated residues in Carcharodon Y 1 R to the corresponding hY 1 R does not rescue their ability to respond to the NPY.From an evolutionary perspective, the reason is that the protein conformation of Carcharodon Y 1 R has undergone significant changes, and rescuing their ability to respond to NPY may require more than a few point mutations (Figures 3E  and S7B-C).Taken together, our results indicated that the positive selection pressure in Osteichthyes Y 1 R maintains the sub-pocket and contributes to retaining the Gi signaling.In contrast, Chondrichthyes were not constrained by the selection pressure, and the mutations in the sub-pocket amino acids led to changes in their Gi signaling.

Conserved common site for NPY recognition by NPYRs
Overall, the NPY-bound Y 1 R and Y 2 R structures shared a similar conformation and central residues, E15-L31 (αhelix region) of NPY formed approximately five α-helical turns and the base of the NPY α-helix overlay in the two structures, while the amino terminus of the Y 1 R helix was rotated approximately 10˚inwards compared with that in Y 2 R (Figure S8A).The residue R 25 of NPY in the Y 1 R structure formed hydrogen bonds with D104 2.68 , which could not be observed in the NPY-Y 2 R structure, potentially contributing to the inwards shift of the NPY-bound Y 1 R α-helix (Figure S8B).In addition, the hydrogen bonds between the residue R 25 of NPY and D104 2.68 further stabilized the NPY-Y 1 R interactions.Consistent with these structural observations, a D104A 2.68 substitution reduced the potency of the Y 1 R response for NPY (∼73 folds) (Figure S8C).NPY in the Y 1 R and Y 2 R complex structures adopted similar conformations in the recognition by the NPYR orthosteric pockets (Figures 4A-D).The C-terminus of NPY (residues 33−36), which was confirmed to display major importance for all NPYR bindings, 3,42 adopted an extended conformation and reaches far into the cores of Y 1 R and Y 2 R, contacting all TM helices except for TM1, as well as residues in ECL2 and ECL3 through an extensive interface of hydrophobic and polar interactions  4A-D).The NPY C-terminal-bound Y 1 R structure exhibited both important common characteristics and notably distinct features compared to the NPY-bound Y 2 R structure (Figures 4A-D).The structural comparison revealed that the Y 1 R-bound NPY side chains R 35 and Y 36 overlaid well with those of Y 2 R-bound NPY, while the NPY residues R 33 and Q 34 occupied different binding sites to those of Y 2 R-bound NPY (Figure 4E).
At the bottom region of the orthosteric peptide-binding pocket, polar receptor residues formed an extensive polar interaction network with amidated Y 36 both in the Y 1 R-and Y 2 R-bound NPY structures, structurally supporting the fact that this amidation modification was necessary for the NPYR activity. 43,44The Y 36 amidation group and hydrogen bond established polar contacts with residues T107 2.61 and S223 5.46 in the NPY-Y 2 R structure (Figures 4C and D), whereas the amidation and carbonyl groups of Y 36 in the Y 1 R-bound NPY formed hydrogen bonds with residues Q120 3.32 and H306 7.39 , respectively (Figures 4A and B).In addition, residue Y 36 was further coordinated by Q219 5.46 through polar interactions and C93 2.57 through van der Waals contacts in the NPY-Y 1 R structure (Figures 4A and B).On the same Y 36 orientation, residue R 35 was fastened mainly through polar interactions by D 6.59 in the NPY-Y 1 R and NPY-Y 2 R structures (Figures 4A-D).Notably, previous studies reported that the ionic interaction of residue D 6.59 is key for positively charged ligand recognition. 45o further identify the common binding sites NPYRs, we conducted a series of mutagenesis analyses.Our NPYR sequence alignments indicated that residues (C 2.57 , T 2.61 , Q 3.32 , and D 6.59 ) in TM2, TM3, and TM6, which form polar interactions and van der Waals contacts with the C-terminus residues Y 36 and R 35 , were conserved among the four receptor subtypes (Figures 4E and F).Mutation of these conserved residues (C 2.57 A, T 2.61 A, Q 3.32 A, and D 6. S2), supporting the essential role of these residues in NPY recognition.Taken together, the structural observations and mutagenesis analyses revealed that NPYRs share common binding sites to bind NPY residues Y 36 and R 35 and adopt different molecular patterns in the interaction with NPY.

2.5
The different recognition patterns in Y 1 R and Y 2 R Our cAMP assay data demonstrated [Leu 31 , Pro 34 ]-NPY retained similar activation potency as NPY on Y 1 R, but displayed higher selectivity for Y 1 R over Y 2 R, indicating that both Y 1 R and Y 2 R has different mechanisms for ligand recognition.Our structures of Y 1 R and Y 2 R in complex with NPY or [Leu 31 , Pro 34 ]-NPY offer templates to understand the ligand recognition or selectivity.In the case of the NPY-Y 1 R structure, the residue Q 34 is observed to interact with only T97 2.61 in Y 1 R through hydrogen bonding (Figure 5A).Whereas the side chain of Q 34 in Y 2 R displayed a different conformation and was projected into a cavity shaped by TM2 and TM7, interacting with T107 2.61 , Y110 2.64 , T111 2.65 , and F307 7.35 in Y 2 R (Figures 5B  and S13A).
To further analyze the selectivity of [Leu 31 , Pro 34 ]-NPY, we determined the structure of the [Leu 31 , Pro 34 ]-NPY-bound Y 1 R in complex with the Gi2 protein.The C-terminal parts of NPY and [Leu 31 , Pro 34 ]-NPY adopted similar binding pose in the orthosteric pocket of Y 1 R (Figure S13B).Residue P 34 of [Leu 31 , Pro 34 ]-NPY share a similar position with residue Q 34 of NPY (Figures 5C and  S13B and C).The substitution of Q with P at position 34 of NPY disrupted the interactions between Q34 and T97 2.61 in Y 1 R.The residue P 34 in [Leu 31 , Pro 34 ]-NPY peptide was not observed to make polar interaction with Y 1 R, and this kind of peptide did not influence Y 1 R signaling activation.Comparing to structures of Y 1 R bound NPY or [Leu 31 , Pro 34 ]-NPY, the residue Q 34 of NPY makes hydrogen bonds with T107 2.61 and T111 2.65 in Y 2 R (Figure 5C).These residues T107 2.61 , Y110 2.64 , T111 2.65 , and F307 7.35 that involved in ligand binding pocket are conserved in both Y 1 R and Y 2 R. We next investigated whether these conserved residues play different roles in Y 1 R and Y 2 R, the residues T 2.61 , Y 2.64 , T 2.65 , and F 7.35 were replaced with Ala both in Y 1 R or Y 2 R. The results of our functional assays showed that all mutants of Y 2 R significant lost NPY-induced signal transduction, which further confirm the important roles of these residues in Y 2 R (Figure S13D).By contrast, T 2.65 A and F 7.35 A mutations in Y 1 R displayed similar activation potency as wild-type receptor in response to NPY (Figure S13D).5D).However, both NPY analogs exhibited different activation potencies for Y 2 R and its mutants (Figure 5E).
In addition, structural comparisons of Y 1 R and Y 2 R revealed a notable displacement of R 33 from NPY peptides in two receptors (Figure 5F).In Y 2 R structure, the residue R 33 is observed to interact with D292 6.59 and forms polar interactions with R 35 and E205 ECL2 via hydrogen bonding (Figure 5F).In Y 1 R structure, the side chain of R 33 in NPY is found to insert a cavity shaped by TM6 and TM7, establishing hydrophobic contacts with residues F302 7.35 , H298 7.31 , and F286 6.58 in Y 1 R (Figure 5F).Consistent with the structural observations, the results of mutagenesis studies and functional assays showed that the mutation E205 ECL2 A influenced NPY induced Y 2 R activation potency, whereas the mutation N283 6.55 A reduced the Y 1 R activation induced by NPY (Figures 5H, S9-12 and Table S2).Notably, the residues F286 6.58 in Y 1 R appears to be a key facet to shape the sub-pocket (Figure 5G).

Structural basis of NPY mediated Y 1 R and Y 2 R activation
The structural comparison of our two Gi2-coupled NPY receptors with other class A GPCRs revealed similar conformations. 46TM6 and TM7 of Y 1 R and Y 2 R adopted nearly identical conformations to the active structures of the neurotensin receptor NTS1, 47 orexin receptors, 35 and the endothelin ET B receptor 33 (Figure S14).Furthermore, the structural comparison of two Y 1 R and Y 2 R receptors with the antagonist-bound NPYRs (PDB ID: 5ZBH, 5ZBQ, and 7DDZ), 31,32 supports the contention that these two complexes were in the active state (Figures 6A and D).Compared with structure of antagonist BMS-193885 bound Y 1 R (PDB ID: 5ZBH), the activated Y 1 R complex displayed pronounced outward displacements (∼8 Å, measured at Cα of T258 6.30 ) of the TM6 at cytoplasmic region, and ∼5 Å inward shift of TM7 (measured at Cα of Y320 7.53 ) (Figure 6A).
The residue F286 6.58 in Y 1 R was demonstrated to play important role in receptor activation as well as ligand selectivity.The detailed comparison of both active and inactive structures reveals a significant movement of F286 6.58 toward receptor helical core, which may initiate the cascade of conformational changes upon agonist binding.Meanwhile, the microswitch residue W 6.48 is observed to display rotameric change (Figures 6C and F), synergistically, F 6.44 in P-I-F motif underwent conformational changes to facilitate G-protein coupling (Figure S15).In addition, residue Q 3.32 conserved in Y 1 R and Y 2 R formed hydrogen bonds with NPY, leading to a slight upward movement of TM3 (Figures 6A, B, D, and E).

DISCUSSION
NPY serves a critical role in modulating a variety of physiological processes in both the central nervous system and peripheral tissues.It exerts its effects through binding to G protein-coupled NPY receptors, with the Y 1 and Y 2 receptor subtypes being particularly noteworthy.Gaining a comprehensive understanding of the structural mechanisms through which Y 1 R and Y 2 R interact with NPY is indispensable for the rational design of selective drugs.
Although it reported some structures of the NPYbound receptor complex 48,49 during the preparation of our manuscript, some new insights about ligand selectivity mechanisms of NPYRs were still obtained in our research.Here, we present three cryo-EM structures of Gi2-coupled Y 1 R and Y 2 R bound to either NPY or [Leu 31 , Pro 34 ]-NPY.These structures reveal a conserved orthosteric peptidebinding pocket in both Y 1 R and Y 2 R that interacts with the C-terminal region of NPY.The extreme C-terminal dipeptide with amidated modification (R 35 -Y 36 -NH 2 ) is buried in the bottom of orthosteric pocket, which is highly conserved between the two NPYR subtypes.Through a combination of structural observations and alanine mutagenesis analysis, we demonstrate that the binding of the NPY Cterminus is critical for the activation potency or efficacy of NPY.
In contrast, distinct physiochemical environments surrounding a tetrapeptide (Y 1 -P 2 -R 33 -Q 34 ) between Y 1 R and Y 2 R serve as determinants for two NPYR subtype preference.Intriguingly, we identified a distinct sub-pocket in Y 1 R that contributes to ligand selectivity and the sub-pocket of Y 1 R plays an essential role in the recognition of the N-terminus of NPY and receptor activation.Notably, Y 1 -P 2 -R 33 forms a specific interaction network with hydrophobic residues F302 7.35 , H298 7.31 , F286 6.58 , enhancing the Y 1 R selectivity.
Moreover, comparisons of the NPY-Y 1 R, NPY-Y 2 R, and [Leu 31 , Pro 34 ]-NPY-Y 1 R complex structures highlight the plasticity of orthosteric pockets across receptor subtypes.Substituting Q 34 with P 34 disrupts the original polar and hydrophobic interactions with Y 2 R, which plays a more indispensable role of Y 2 R to interact with NPY than Y 1 R, thus leading to the Y 2 R selectivity.
In summary, we offer detailed molecular maps depicting the binding of NPY peptides to various NPY receptor subtypes, thereby shedding light on subtype-specific interaction patterns.This critical insight substantially broadens (B) Conformational changes comprising of F286 6.58 , H298 7.31 , and F302 7.35 upon peptide activation.UR-MK299 push the side-chain of F286 6.58 to swing away from the receptor helical core due to steric clash.The swing orientations of F286 6.58 , H298 7.31 , and F302 7.35  our knowledge of ligand recognition and signal transduction within the NPY-GPCR system.Consequently, our findings establish a robust foundation for the future development of selective drugs aimed at specific NPY receptor targets, thereby paving the way for more precise and efficacious therapeutic strategies.

MATERIALS AND METHODS
The details of the protein expression and purification, cryo-EM structure determination, and pharmacological experiments are provided in Supplementary Information Methods.

A U T H O R C O N T R I B U T I O
complexes.(A) Sequence alignment of different N-terminally truncated NPY and [Leu 31 , Pro 34 ]-NPY.The alignment was generated by ESPript3 with CLUSTALW (red squares, identical residues).(B) Concentration-response curves of Y 1 R in response to stimulation with NPY, [Leu

F I G U R E 2
The recognition of N-terminus of NPY by Y 1 R. (A and B) Cut-away view of the ligand-binding pocket of Y 1 R in complex with NPY (top view).The sub-pocket for the N-terminus of NPY is shown in detail (B).(C) and (D) Cut-away view of the ligand-binding pocket of Y 2 R in complex with NPY (top view).The interactions between NPY and Y 2 R are shown in detail (D).(E) Sequence alignment of the residues forming sub-pocket inY 1 R and Y 2 R. (F) Concentration-response curves of Y 1 R and polar residues of sub-pocket mutations in response to stimulation with NPY, respectively.Values are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate.(G) Concentration-response curves of Y 1 R and hydrophobic residues of sub-pocket mutations in response to stimulation with NPY, respectively.Values are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate.

F I G U R E 3
The evolution of Y 1 R in vertebrates.(A) Diagram of Y 1 R in vertebrates and invertebrates.(B) Concentration-response curves of Y 1 R in response to stimulation with NPY in vertebrates.Data represent mean ± s.e.m. from three independent experiments (n = 3) performed in triplicate.(C) The parameters and statistical significance of LRTs for branch of Osteichthyes Y 1 R; the dN/dS ratio is calculated with the whole protein coding region; **, *, and no * indicate p values in excess of 0.99, 0.95, and 0.90, respectively.(D) Sequence logo of Y 1 Rs in vertebrates.Red triangles represent positions of sub-pocket sites of Y 1 Rs.(E) Representative of effects of hY1R mutations, drY1R mutations, and ccY1R mutations of the Y 1 R on NPY in cAMP accumulation assays.Data represent mean ± s.e.m. from three independent experiments (n = 3) performed in triplicate.

F I G U R E 4
Conserved common sites for C-terminus of NPY recognition by NPYRs.(A) Ligand-binding pocket for the C-terminus of NPY.Y 1 R is shown in cyan cartoon representation.The Y 1 R residues that form interactions with the C-terminus of NPY are shown as sticks.The C-terminus residues (R 33 -Q 34 -R 35 -Y 36 -NH 2 ) of NPY (carbon in orange) is shown as sticks and hydrogen bonds are shown as black dashed lines.(B) Schematic representation of interactions between Y 1 R and the C-terminus residues (R 33 -Q 34 -R 35 -Y 36 -NH 2 ) of NPY analyzed using LigPlot+ program. 50The Y 1 R residues engaged in hydrogen bonds are shown as cyan sticks.Hydrogen bonds are shown as black dashed lines.(C) Ligand-binding pocket for the C-terminus of NPY.Y 2 R is shown in medium slate cartoon representation.The Y 2 R residues that form interactions with the C-terminus of NPY are shown as sticks.The C-terminus residues (R 33 -Q 34 -R 35 -Y 36 -NH 2 ) of NPY (carbon in magenta) is shown as sticks and hydrogen bonds are shown as red dashed lines.(D) Schematic representation of interactions between Y 2 R and the (Figures 59 A) in Y 1 R, Y 2 R, Y 4 R, and Y 5 R significantly impaired receptor activity (Figures 4G, S9-12 and Table

C
-terminus residues (R33 -Q34 -R35 -Y36 -NH 2 ) of NPY analyzed using LigPlot+ program.The Y 2 R residues engaged in hydrogen bonds are shown as medium slate sticks.Hydrogen bonds are shown as red dashed lines.(E) Comparison of C-terminus of NPY binding modes between Y 2 R and Y 1 R. (F) Sequence alignment of the conserved residues for C-terminus of NPY recognition in Y 1 R and Y 2 R. (G) NPY-induced cAMP accumulation assays of the conserved common sites in Y 1 R, Y 2 R, Y 4 R, and Y 5 R. Bars represent differences in calculated NPY potency [pEC 50 ] for each mutant relative to the wild-type receptor (WT).Values are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate.P Values were determined by one-way of variance ANOVA with Dunnett's test.*p < 0.05; **p < 0.01; ***p < 0.001; ****p ≤ 0.0001; ns, no significant difference.

F I G U R E 5
The selectivity for NPY and [Leu 31 , Pro 34 ]-NPY recognitions by Y 1 R and Y 2 R. (A) Detailed interactions between C-terminus residues Q 34 (sticks, orange) of NPY and Y 1 R (cyan) are shown.The residues interacting with Q 34 of Y 2 R are shown as sticks and surface.The polar contacts are shown as black dashed lines.(B) Detailed interactions between C-terminus residues Q 34 (sticks, magenta) of NPY and Y 2 R (medium slate) are shown.The residues interacting with Q 34 of Y 2 R are shown as sticks and surface.The polar contacts are shown as red dashed lines.(C) Comparison of the detailed interactions between C-terminus residues Q 34 (sticks, orange, magenta) of NPY or P 34 of [Leu 31 , Pro 34 ]-NPY (sticks, light salmon) and residues of Y 1 R and Y 2 R. (D and E) NPY-induced or [Leu 31 , Pro 34 ]-NPY-induced cAMP accumulation assays of the sites in Y 1 R (D) and Y 2 R (E).Bars represent differences in calculated NPY or [Leu 31 , Pro 34 ]-NPY potency [pEC 50 ] for each mutant relative to the wild-type receptor (WT).Values are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate.p Values were determined by unpaired t-test.**p < 0.01; ***p < 0.001; ****p ≤ 0.0001; ns, no significant difference.(F) Comparison of the detailed interactions between C-terminus residues R 33 (sticks, orange, magenta) of NPY and residues (sticks, cyan, medium slate) of Y 1 R and Y 2 R. The residues interacting with Q 33 of Y 1 R and Y 2 R are shown as sticks and surface.(G) The detailed interactions between C-terminus residues R 33 (sticks, orange) of NPY and residues (sticks, cyan) of Y 1 R. (H) NPY-induced cAMP accumulation assays of the sites in Y 1 R and Y 2 R. Bars represent differences in calculated NPY potency [pEC 50 ] for each mutant relative to the wild-type receptor (WT).Values are shown as the mean ± s.e.m. of three experiments (n = 3) performed in triplicate.p Values were determined by unpaired t-test.**p < 0.01; ***p < 0.001; ****p ≤ 0.0001.To further compare the pharmacological featured of NPY and [Leu 31 , Pro 34 ]-NPY, we measured the NPY or [Leu 31 , Pro 34 ]-NPY-induced Y 1 R and Y 2 R activation.For Y 1 R, NPY and [Leu 31 , Pro 34 ]-NPY displayed similar potencies on Y 1 R or mutants (Figure

F I G U R E 6
Mechanism of Y 1 R and Y 2 R activation by NPY binding.(A) Structural superposition of the active and antagonist-bound Y 1 R.
were indicated by red arrows.The clashes were highlighted as red oval dashed lines.(C) Conformational changes of Toggle switch of the conserved "micro-switches" upon Y 1 R activation.The conformational changes of residue side chains and the outward displacement of TM6 are shown as red arrows upon receptor activation.(D) Structural superposition of the active and antagonist-bound Y 2 R. (E) Conformational changes comprising of residues interacting with NPY upon peptide activation.D 6.59 displays upon movement, shown as a black arrow.(F) Conformational changes of the toggle switch in conserved "micro-switches" upon receptor activation.The conformational alterations of residue side chains are depicted with red arrows following receptor activation.Additionally, the outward displacement of TM6 in the active receptor is illustrated by a red arrow.
N S W. Y. and Z. S. initiated structural studies of NPYRs and their ligands.S. S. and C. S. designed the expression constructs, purification, and preparation of the NPY-Y 1 R-Gi2, NPY-Y 2 R-Gi2 and [Leu 31 , Pro 34 ] NPY-Y 1 R-Gi2 complexes.S. S., L. C., and C. Z. carried out cryo-EM screening, data collection, and model building and refinement in the study.Y. D., C. W., and K. W. performed functional assays.Z. Y. and H. H. contributed in purification of scFv16.H. C. performs bioinformatics analysis under the direction of C. D. S. S. and Y. D. prepared figures.S. S., C. S., and Y. D. planned and coordinated the entire project under the supervision of W. Y. and Z. S. W. Y., F. Y., C. D., and Z. S. supervised the overall project and wrote the 31 34